Novel Insights into Interleukin 6 (IL-6) Cis- and Trans-signaling Pathways by Differentially Manipulating the Assembly of the IL-6 Signaling Complex

The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6·IL-6 receptor (IL-6R)·gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound or soluble form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both membrane-bound IL-6R and soluble IL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R but allows the binding of IL-6 to the IL-6R and the recruitment of gp130, forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling, suggesting that the cis- and trans-modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling, although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared with a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different, and this should be taken into account when developing strategies to inhibit IL-6 clinically.     

Protein tyrosine phosphatase non-receptor type 22 (PTPN22) +1858 C>T gene polymorphism in Egyptian cases with rheumatoid arthritis

Background

Rheumatoid arthritis (RA) is a common autoimmune disease with a complex genetic background. The gene encoding protein tyrosine phosphatase non-receptor type 22 (PTPN22) has been reported to be associated with RA in several populations.

Objectives

This work aimed at assessing the association of PTPN22 +1858 C>T gene polymorphism with the susceptibility, activity and severity of RA in Egyptian subjects.

Subjects and methods

This study included 112 unrelated RA patients who were compared to 122 healthy unrelated individuals taken from the same locality. For all subjects, DNA was genotyped for PTPN22 +1858 C>T (rs2476601) polymorphism using the PCR-RFLP technique. Antibodies to cyclic citrullinated peptides (anti-CCP) were measured by enzyme-linked immunosorbent assay (ELISA).

Results

Cases showed significantly higher PTPN22 +1858 T allele carriage rate (CT + TT genotypes) compared to controls (34.8% vs. 8.2%, OR = 5.98, 95% CI = 2.81–12.73, p < 0.001). Also the frequency of the PTPN22 +1858 T allele was significantly higher among cases compared to controls (18.7% vs. 4.5%, OR = 4.89; 95% CI = 2.45–9.76, p < 0.001). Cases positive to the PTPN22 T allele (CT + TT genotypes) showed no significant difference from those with the CC genotype regarding clinical and immune parameters. Nonetheless, they showed a more functional disability presented in their significantly higher health assessment questionnaire (HAQ) score (p = 0.04).

Conclusions

This study is a confirmatory evidence of the association of the PTPN22 +1858 T allele with susceptibility and functional disability of RA in Egyptian subjects.     

Allosteric peptides bind a caspase zymogen and mediate caspase tetramerization

The caspases are a family of cytosolic proteases with essential roles in inflammation and apoptosis. Drug discovery efforts have focused on developing molecules directed against the active sites of caspases, but this approach has proved challenging and has not yielded any approved therapeutics. Here we describe a new strategy for generating inhibitors of caspase-6, a potential therapeutic target in neurodegenerative disorders, by screening against its zymogen form. Using phage display to discover molecules that bind the zymogen, we report the identification of a peptide that specifically impairs the function of caspase-6 in vitro and in neuronal cells. Remarkably, the peptide binds at a tetramerization interface that is uniquely present in zymogen caspase-6, rather than binding into the active site, and acts via a new allosteric mechanism that promotes caspase tetramerization. Our data illustrate that screening against the zymogen holds promise as an approach for targeting caspases in drug discovery.     

Effect of in utero exposure to diethylstilbestrol on lumbar and femoral bone, articular cartilage, and the intervertebral disc in male and female adult mice progeny with and without swimming exercise

Introduction

Developmental exposure to estrogens has been shown to affect the musculoskeletal system. Furthermore, recent studies have shown that environmental exposure to estrogen-like compounds is much higher than originally anticipated. The aim of this study was to determine the effects of diethylstilbestrol (DES), a well-known estrogen agonist, on articular cartilage, intervertebral disc (IVD), and bone phenotype.

Methods

C57Bl/6 pregnant mice were dosed orally with vehicle (peanut oil) or 0.1, 1.0, and 10 μg/kg/day of DES on gestational days 11 to 14. Male and female pups were allowed to mature without further treatment until 3 months of age, when swim and sedentary groups were formed. After euthanasia, bone mineral density (BMD), bone mineral content (BMC), bone area (BA), and trabecular bone area (TBA) of the lumbar vertebrae and femur were measured by using a PIXImus Bone Densitometer System. Intervertebral disc proteoglycan was measured with the DMMB assay. Histologic analysis of proteoglycan for IVD and articular cartilage was performed with safranin O staining, and degeneration parameters were scored.

Results

The lumbar BMC was significantly increased in female swimmers at both the highest and lowest dose of DES, whereas the femoral BMC was increased only at the highest. The males, conversely, showed a decreased BMC at the highest dose of DES for both lumbar and femoral bone. The female swim group had an increased BA at the highest dose of DES, whereas the male counterpart showed a decreased BA for femoral bone. The TBA showed a similar pattern. Proteoglycan analysis of lumbar IVDs showed a decrease at the lowest doses but a significant increase at the highest doses for both males and females. Histologic examination showed morphologic changes of the IVD and articular cartilage for all doses of DES.

Conclusions

DES significantly affected the musculoskeletal system of adult mice. Results suggest that environmental estrogen contaminants can have a detrimental effect on the developmental lumbar bone growth and mineralization in mice. Further studies measuring the impact of environmental estrogen mimics, such as bisphenol A, are then warranted.     

Modeling and Statistical Analysis of the Spatio-Temporal Patterns of Seasonal Influenza in Israel

Background

Seasonal influenza outbreaks are a serious burden for public health worldwide and cause morbidity to millions of people each year. In the temperate zone influenza is predominantly seasonal, with epidemics occurring every winter, but the severity of the outbreaks vary substantially between years. In this study we used a highly detailed database, which gave us both temporal and spatial information of influenza dynamics in Israel in the years 1998–2009. We use a discrete-time stochastic epidemic SIR model to find estimates and credible confidence intervals of key epidemiological parameters.

Findings

Despite the biological complexity of the disease we found that a simple SIR-type model can be fitted successfully to the seasonal influenza data. This was true at both the national levels and at the scale of single cities.The effective reproductive number R_e varies between the different years both nationally and among Israeli cities. However, we did not find differences in R_e between different Israeli cities within a year. R _e was positively correlated to the strength of the spatial synchronization in Israel. For those years in which the disease was more “infectious”, then outbreaks in different cities tended to occur with smaller time lags. Our spatial analysis demonstrates that both the timing and the strength of the outbreak within a year are highly synchronized between the Israeli cities. We extend the spatial analysis to demonstrate the existence of high synchrony between Israeli and French influenza outbreaks.

Conclusions

The data analysis combined with mathematical modeling provided a better understanding of the spatio-temporal and synchronization dynamics of influenza in Israel and between Israel and France. Altogether, we show that despite major differences in demography and weather conditions intra-annual influenza epidemics are tightly synchronized in both their timing and magnitude, while they may vary greatly between years. The predominance of a similar main strain of influenza, combined with population mixing serve to enhance local and global influenza synchronization within an influenza season.     

A whole cell assay to measure caspase-6 activity by detecting cleavage of lamin A/C

Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context.     

"In vitro" capacitation and subsequent acrosome reaction are related to changes in the expression and location of midpiece actin and mitofusin-2 in boar spermatozoa

The induction of “in vitro” capacitation (IVC) and subsequent, progesterone-induced “in vitro” acrosome reaction (IVAR) was concomitant with an increase in actin polymerization, also showing an increase in actin presence at the apical area of the midpiece. The presence of mitofusin-2, a protein involved in the regulation of the coordinated mitochondrial function, expanded from midpiece to the principal piece after IVC and IVAR. All of these results indicate that the increase of boar sperm mitochondrial activity during IVC and the first minutes of IVAR is concomitant with changes in the expression and location of both actin and mitofusin-2. Our results suggest that both actin and mitofusin-2 play important roles in the modulation of boar sperm mitochondrial function, both by originating changes in the protein membrane environment and by changes in the mitochondrial structure itself.     

A bacterial reporter panel for the detection and classification of antibiotic substances

The ever‐growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy‐based non‐antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.     

WW-domain-containing oxidoreductase is associated with low plasma HDL-C levels

Low serum HDL-cholesterol (HDL-C) is a major risk factor for coronary artery disease. We performed targeted genotyping of a 12.4 Mb linked region on 16q to test for association with low HDL-C by using a regional-tag SNP strategy. We identified one SNP, rs2548861, in the WW-domain-containing oxidoreductase (WWOX) gene with region-wide significance for low HDL-C in dyslipidemic families of Mexican and European descent and in low-HDL-C cases and controls of European descent (p = 6.9 × 10⁻⁷). We extended our investigation to the population level by using two independent unascertained population-based Finnish cohorts, the cross-sectional METSIM cohort of 4,463 males and the prospective Young Finns cohort of 2,265 subjects. The combined analysis provided p = 4 × 10⁻⁴ to 2 × 10⁻⁵. Importantly, in the prospective cohort, we observed a significant longitudinal association of rs2548861 with HDL-C levels obtained at four different time points over 21 years (p = 0.003), and the T risk allele explained 1.5% of the variance in HDL-C levels. The rs2548861 resides in a highly conserved region in intron 8 of WWOX. Results from our in vitro reporter assay and electrophoretic mobility-shift assay demonstrate that this region functions as a cis-regulatory element whose associated rs2548861 SNP has a specific allelic effect and that the region forms an allele-specific DNA-nuclear-factor complex. In conclusion, analyses of 9,798 subjects show significant association between HDL-C and a WWOX variant with an allele-specific cis-regulatory function.